Food and waterborne protozoal infections are a common cause of severe human disease worldwide. The Apicomplexa consist of numerous protozoa which cause disease, including Plasmodium, Toxoplasma, Cryptosporidia, and Babesia. Toxoplasma gondii is a Category B Priority pathogen which has an excellent small animal model, and is amenable to genetic manipulation. Oral infection with T. gondii in susceptible mouse strains results in inflammation and ileitis. In studies evaluating innate immunity to T. gondii, we have identified the signaling adaptor protein, DAP10, as important in protecting mice from lethal infection, toxoplasma replication, and ileitis. DAP10 is the signaling adaptor for the activating immunoreceptor NKG2D, which recognizes ligands on stressed, infected and transformed cells. Mice deficient in DAP10 express NKG2D on NK cells, but not on CD8+ T cells or gamma/delta-T cells. These mice are more susceptible to oral challenge with T. gondii. DAP10-/- mice have more severe intestinal inflammation and necrosis, higher parasite burdens in the spleen, and succumb to infection at doses of T. gondii which wild type mice routinely survive. Studies outlined in this proposal aim to determine whether the increased susceptibility of DAP10-/- mice is due to increased replication of T. gondii in the intestine due to lack of immune response by DAP10 expressing CD8+ T cells, macrophages and dendritic cells, or due to intestinal necrosis secondary to the loss of NKG2D/DAP10 mediated regulatory function of CD8+ T cells in the intestine. We will also directly test the hypothesis that the increased susceptibility in DAP10-/- mice is due to lack of NKG2D signaling. These studies will further delineate the innate immune response to oral challenge with T. gondii, and provide important insights to apicomplexan immunology as well as mucosal immunity to oral pathogens. Relevance to public health: Toxoplasma gondii is a common and important human pathogen, and is a model for other protozoal pathogens, including malaria. We have identified the activating immune receptor, NKG2D, as important for the natural, innate immune response to pathogens. Studies in this grant aim to define its role in oral infection with T. gondii.